Carcinogenic Aristolochic Acid Research Articles

Exceptionally long-term persistence of DNA adducts formed by carcinogenic aristolochic acid I in renal tissue from patients with aristolochic acid nephropathy.

Aristolochic acid (AA) causes aristolochic acid nephropathy (AAN), first described in women in Belgium accidently prescribed Aristolochia fangchi in a slimming treatment, and also Balkan endemic nephropathy (BEN), through probable dietary contamination with Aristolochia clematitis seeds. Both nephropathies have a high risk of urothelial cancer, with AA being the causative agent. In tissues of AAN and BEN patients, a distinct DNA adduct, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), has been detected. DNA adducts can be removed through DNA repair, they can result in mutations through erroneous DNA replication or they can cause cell death. The dA-AAI adduct induces AT to TA transversions in the tumor-suppressor TP53 gene in experimental systems, matching TP53 mutations observed in urothelial tumors from AAN cancer cases. Using thin-layer chromatography 32P-postlabeling and mass spectrometric analysis we report the detection of dA-AAI in renal DNA from 11 Belgian AAN patients over 20 years after exposure to AA had ceased. Our results showed that dA-AAI is an established biomarker of AA exposure, and that this biomarker can be demonstrated to be persistent decades after a distinct AA exposure. Further, the persistence of dA-AAI adducts appears to be a critical determinant for the AA mutational fingerprint frequently found in oncogenes and tumor suppressor genes recently identified by whole genome sequencing of AA-associated urothelial tumors. The potential for exposure to AA worldwide is high; the unprecedented long-term persistence of dA-AAI provides a useful long-term biomarker of exposure and attests to the role of AA in human urothelial malignancy.

Molecular identification and cytotoxicity study of herbal medicinal materials that are confused by Aristolochia herbs

Herbal materials derived from Aristolochia species contain carcinogenic aristolochic acids (AAs) and have been used as traditional Chinese medicines (TCMs) or adulterants of other TCMs. The purpose of this study is to identify the TCMs Stephaniae Tetrandrae Radix, Akebiae Caulis, Aucklandia Radix and Aristolochiae Fructus by sequencing the matK, rbcL, trnH-psbA and trnL-trnF DNA regions. The cytotoxicities of AAs and these TCMs were also studied in COS-7 and HEK-293 cells. Diagnostic polymorphic sites were identified in all the four DNA loci for the differentiation of genuine herbs from their adulterants/substitute. The 48h IC50 of AAI were 78μM (COS-7) and 70μM (HEK-293) while the IC50 of AAII were higher than 100μM in both cell lines. Except Aucklandia Radix, cytotoxicity study also showed that AA-containing herbs were more toxic than their corresponding genuine herbs and substitute.

Genome-Wide Mutational Signatures of Aristolochic Acid and Its Application as a Screening Tool

Aristolochic acid (AA), a natural product of Aristolochia plants found in herbal remedies and health supplements, is a group 1 carcinogen that can cause nephrotoxicity and upper urinary tract urothelial cell carcinoma (UTUC). Whole-genome and exome analysis of nine AA-associated UTUCs revealed a strikingly high somatic mutation rate (150 mutations/Mb), exceeding smoking-associated lung cancer (8 mutations/Mb) and ultraviolet radiation-associated melanoma (111 mutations/Mb). The AA-UTUC mutational signature was characterized by A:T to T:A transversions at the sequence motif A[C|T]AGG, located primarily on nontranscribed strands. AA-induced mutations were also significantly enriched at splice sites, suggesting a role for splice-site mutations in UTUC pathogenesis. RNA sequencing of AA-UTUC confirmed a general up-regulation of nonsense-mediated decay machinery components and aberrant splicing events associated with splice-site mutations. We observed a high frequency of somatic mutations in chromatin modifiers, particularly KDM6A, in AA-UTUC, demonstrated the sufficiency of AA to induce renal dysplasia in mice, and reproduced the AA mutational signature in experimentally treated human renal tubular cells. Finally, exploring other malignancies that were not known to be associated with AA, we screened 93 hepatocellular carcinoma genomes/exomes and identified AA-like mutational signatures in 11. Our study highlights an unusual genome-wide AA mutational signature and the potential use of mutation signatures as "molecular fingerprints" for interrogating high-throughput cancer genome data to infer previous carcinogen exposures.

A Novel p53 Mutant Found in Iatrogenic Urothelial Cancers Is Dysfunctional and Can Be Rescued by a Second-site Global Suppressor Mutation

Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A→T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.

NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.

Bioactivation versus Detoxication of the Urothelial Carcinogen Aristolochic Acid I by Human Cytochrome P450 1A1 and 1A2

Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. Individual susceptibility to AA-induced disease likely reflects individual differences in enzymes that metabolize AA. Herein, we evaluated AAI metabolism by human cytochrome P450 (CYP) 1A1 and 1A2 in two CYP1A-humanized mouse lines that carry functional human CYP1A1 and CYP1A2 genes in the absence of the mouse Cyp1a1/1a2 orthologs. Human and mouse hepatic microsomes and human CYPs were also studied. Human CYP1A1 and 1A2 were found to be principally responsible for reductive activation of AAI to form AAI-DNA adducts and for oxidative detoxication to 8-hydroxyaristolochic acid (AAIa), both in the intact mouse and in microsomes. Overall, AAI-DNA adduct levels were higher in CYP1A-humanized mice relative to wild-type mice, indicating that expression of human CYP1A1 and 1A2 in mice leads to higher AAI bioactivation than in mice containing the mouse CYP1A1 and 1A2 orthologs. Furthermore, an exclusive role of human CYP1A1 and 1A2 in AAI oxidation to AAIa was observed in human liver microsomes under the aerobic (i.e., oxidative) conditions. Because CYP1A2 levels in human liver are at least 100-fold greater than those of CYP1A1 and there exists a > 60-fold genetic variation in CYP1A2 levels in human populations, the role of CYP1A2 in AAI metabolism is clinically relevant. The results suggest that, in addition to CYP1A1 and 1A2 expression levels, in vivo oxygen concentration in specific tissues might affect the balance between AAI nitroreduction and demethylation, which in turn would influence tissue-specific toxicity or carcinogenicity.

The human carcinogen aristolochic acid i is activated to form DNA adducts by human NAD(P)H:quinone oxidoreductase without the contribution of acetyltransferases or sulfotransferases

Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with AA nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. We investigated the efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) to activate aristolochic acid I (AAI) and used in silico docking, using soft-soft (flexible) docking procedure, to study the interactions of AAI with the active site of human NQO1. AAI binds to the active site of NQO1 indicating that the binding orientation allows for direct hydride transfer (i.e., two electron reductions) to the nitro group of AAI. NQO1 activated AAI, generating DNA adduct patterns reproducing those found in urothelial tissues from humans exposed to AA. Because reduced aromatic nitro-compounds are often further activated by sulfotransferases (SULTs) or N,O-acetlytransferases (NATs), their roles in AAI activation were investigated. Our results indicate that phase II reactions do not play a major role in AAI bioactivation; neither native enzymes present in human hepatic or renal cytosols nor human SULT1A1, -1A2, -1A3, -1E, or -2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAI. Instead under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAI through the formation of a cyclic hydroxamic acid (N-hydroxyaristolactam I) favored by the carboxy group in peri position to the nitro group without additional conjugation. These results emphasize the major importance of NQO1 in the metabolic activation of AAI and provide the first evidence that initial nitroreduction is the rate limiting step in AAI activation.

Identification of a reduction product of aristolochic acid: implications for the metabolic activation of carcinogenic aristolochic acid.

Aristolochic acids are nephrotoxic and carcinogenic natural products that have been implicated both in endemic nephropathy in the Balkan region and in ailments caused by ingestion of herbal remedies. Aristolochic acids are metabolized to active intermediates that bind to DNA. In this study, reduction of aristolochic acid I with zinc in acetic acid afforded a new product that was characterized as 9-methoxy-7-methyl-2H-1,3-oxazolo[5',4'-10,9]phenanthro[3,4-d]-1,3-dioxolane-5-carboxylic acid, designated as aristoxazole, along with the expected aristolactam I. This new compound is a condensation product of aristolochic acid and acetic acid that may be related to the aristolochic acid-DNA adducts. The proposed mechanism of formation of aristoxazole involves nucleophilic attack of acetic acid on the nitrenium ion of aristolochic acid I. On the basis of these studies, a route to the metabolic activation of aristolochic acids and formation of adducts with DNA in in vitro systems is proposed and discussed.

A novel and specific method for the determination of aristolochic acid-derived DNA adducts in exfoliated urothelial cells by using ultra performance liquid chromatography–triple quadrupole mass spectrometry

Aristolochic acid nephropathy (AAN) is associated with the prolonged exposure to nephrotoxic and carcinogenic aristolochic acids (AAs). DNA adducts induced by AAs have been proven to be critical biomarkers for AAN. Therefore, accurate and specific quantification of AA–DNA adducts is important. In this study, a specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and applied for the determination of 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI) in exfoliated urothelial cells of AA-dosed rats. After the isolation from urine samples, DNA in urothelial cells were subjected to enzymatic digestion and solid-phase extraction on a C18 Sep-Pak cartridge for the enrichment of DNA adducts. The sample extracts were analyzed by reverse-phase UPLC–MS/MS with electrospray ionization in positive ion mode. The quantification of the AA–DNA adduct was performed by using multiple reaction monitoring with reserpine as internal standard. The method provided good accuracy and precision with a detection limit of 1ng/ml, which allowed the detection of trace of dA-AAI in exfoliated urothelial cells. After one-month oral dose of AAI at 10mg/kg/day, 2.1±0.3 dA-AAI per 109 normal dA was detected in exfoliated urothelial cells of rats. Compared to the traditional methods such as 32P-postlabelling and HPLC with fluorescence detection, the developed UPLC–MS/MS method is more specific and rapid with a retention time of 4min. The outcome of this study may have clinical significance for diagnosing and monitoring AA-associated disease because detection of DNA adducts in exfoliated urothelial cells is non-invasive and convenient.

Gene expression changes induced by the human carcinogen aristolochic acid I in renal and hepatic tissue of mice

Aristolochic acid (AA) is the causative agent of urothelial tumors associated with AA nephropathy and is also implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. These tumors contain AA-characteristic TP53 mutations. We examined gene expression changes in Hupki (human TP53 knock-in) mice after treatment with aristolochic acid I (AAI) by gavage (5 mg/kg body weight). After 3, 12 and 21 days of treatment gene expression profiles were investigated using Agilent Whole Mouse 44K Genome Oligo Array. Expression profiles were significantly altered by AAI treatment in both target (kidney) and nontarget (liver) tissue. Renal pathology and DNA adduct analysis confirmed kidney as the target tissue of AAI-induced toxicity. Gene ontology for functional analysis revealed that processes related to apoptosis, cell cycle, stress response, immune system, inflammatory response and kidney development were altered in kidney. Canonical pathway analysis indicated Nfκb, aryl hydrocarbon receptor, Tp53 and cell cycle signaling as the most important pathways modulated in kidney. Expression of Nfκb1 and other Nfκb-target genes was confirmed by quantitative real-time PCR (qRT-PCR) and was consistent with the induction of Nfκb1 protein. Myc oncogene, frequently overexpressed in urothelial tumors, was upregulated by AAI on the microarrays and confirmed by qRT-PCR and protein induction. Collectively we found that microarray gene expression analysis is a useful tool to define tissue-specific responses in AAI-induced toxicity. Several genes identified such as TP53, Rb1, Mdm2, Cdkn2a and Myc are frequently affected in human urothelial cancer, and may be valuable prognostic markers in future clinical studies.

A new approach for the sensitive determination of DNA adduct of aristolochic acid II by using high-performance liquid chromatography with fluorescence detection

A sensitive high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for the determination of DNA adducts induced by nephrotoxic and carcinogenic aristolochic acid (AA) is presented. The DNA adduct of AAII (dA-AAII) was synthesized by in vitro incubation, purified by preparative HPLC, characterized using fluorescence spectroscopy and high-resolution mass spectrometry, and was used as the biomarker for AA exposure in rats. The developed HPLC-FLD method was validated and applied for the determination of dA-AAII in rat kidney tissues. The method provided a detection limit of 18.3 fmol, which allowed the detection of dA-AAII in the rat kidney tissue samples collected after a single oral dose of AA. dA-AAII was detected in the kidney DNA digestion extracts of the rats that were dosed with AA at 5 mg/kg and 30 mg/kg at concentrations of 6.2 ± 1.1 and 41.3 ± 8.0 dA-AAII per 10 9 normal dA, respectively.

Characterization of the DNA adducts induced by aristolochic acids in oligonucleotides by electrospray ionization tandem mass spectrometry

Metabolic activation of carcinogenic aristolochic acids (AA) produces reactive aristolactam-nitrenium ion intermediates. Electrophilic attack of the aristolactam-nitrenium ion via its C7 position to the exocyclic amino group in the purine bases leads to the formation of DNA adducts. DNA-binding assays have demonstrated that carcinogens show site- and sequence-specificity and the biological consequence is defined by the nature of binding as well as their position in the genome. In this study, electrospray ionization tandem mass spectrometry was applied for the identification and position mapping of DNA adducts in oligonucleotides (ODNs). The developed method was successfully applied for the analysis of unmodified and AA-modified ODNs (5'-TTTATT-3', 5'-TTTGTT-3' and 5'-TACATGTGT-3'). The observation of the modified bases (modified adenine and guanine) together with the complementary product ions ([a(n)-B*(n)](-), w(-)) from the cleavage of the 3' C--O bond adjacent to the modified base in MS/MS analyses readily enabled the identification of the AA-binding site in ODNs.

Methodologies for the quality assessment of medicinal plants and phytopharmaceuticals

The abundant consumption of herbal medicines is often associated with toxicity problems due to the presence of pesticide residues, bacterial contaminations and confusions between plant species. Thus besides efficacy, safety represents an important issue in phytotherapy, because many plants are used as dietary supplements. In this respect, different cases of intoxication, mostly renal failure, have been reported after consumption of slimming regimens containing nephrotoxic and carcinogenic aristolochic acids [1]. Hepatotoxicity problems have also been reported for different plants containing pyrrolizidine alkaloids such as Petasites hybridus or Tussilago farfara (Asteraceae) [2]. Other toxicity issues have also been reported for edible plants which can be botanically confused and cases are known in which leaves of the autumn crocus (Colchicum autumnale, Liliaceae) have been consumed instead of ramsons (Allium ursinum, Liliaceae). Ginkgo biloba can also present some safety problems related to its content in allergenic alkylphenols [3]. Such hazardous compounds are present only in very low concentrations in phyto-pharmaceutical preparations and European regulation requires that their content do not exceed 5 ppm. In order to solve some of the issues concerning the quality control and standardisation of plant preparations, analytical strategies based on the use of LC-UV-MS, UPLC-TOF-MS and LC-NMR have been developed for a rapid profiling of the extracts [4]. Different examples of analyses in relation with toxicity assessment and quality control will be discussed. The use of these methods for dereplication purposes in the search of new lead compounds from plants will also be treated.

Gene expression profiles modulated by the human carcinogen aristolochic acid I in human cancer cells and their dependence on TP53

Aristolochic acid (AA) is the causative agent of urothelial tumours associated with aristolochic acid nephropathy. These tumours contain TP53 mutations and over-express TP53. We compared transcriptional and translational responses of two isogenic HCT116 cell lines, one expressing TP53 (p53-WT) and the other with this gene knocked out (p53-null), to treatment with aristolochic acid I (AAI) (50−100 μM) for 6−48 h. Modulation of 118 genes was observed in p53-WT cells and 123 genes in p53-null cells. Some genes, including INSIG1, EGR1, CAV1, LCN2 and CCNG1, were differentially expressed in the two cell lines. CDKN1A was selectively up-regulated in p53-WT cells, leading to accumulation of TP53 and CDKN1A. Apoptotic signalling, measured by caspase-3 and -7 activity, was TP53-dependent. Both cell types accumulated in S phase, suggesting that AAI-DNA adducts interfere with DNA replication, independently of TP53 status. The oncogene MYC, frequently over-expressed in urothelial tumours, was up-regulated by AAI, whereas FOS was down-regulated. Observed modulation of genes involved in endocytosis, e.g. RAB5A, may be relevant to the known inhibition of receptor-mediated endocytosis, an early sign of AA-mediated proximal tubule injury. AAI-DNA adduct formation was significantly greater in p53-WT cells than in p53-null cells. Collectively, phenotypic anchoring of the AAI-induced expression profiles to DNA adduct formation, cell-cycle parameters, TP53 expression and apoptosis identified several genes linked to these biological outcomes, some of which are TP53-dependent. These results strengthen the importance of TP53 in AA-induced cancer, and indicate that other alterations, e.g. to MYC oncogenic pathways, may also contribute.

Liquid chromatography/mass spectrometry for metabonomics investigation of the biochemical effects induced by aristolochic acid in rats: the use of information‐dependent acquisition for biomarker identification

The toxic effects of oral administrations of nephrotoxic and carcinogenic aristolochic acid (AA) to male Sprague-Dawley rats were investigated by using high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Analysis of the urine and plasma samples revealed distinct changes in the biochemical patterns in the AA-dosed rats. After peak finding and alignment, principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for multivariate data analysis. Potential biomarkers were studied by high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses. The MS/MS spectra of all endogenous metabolites satisfying the pre-defined criteria were acquired in a single information-dependent acquisition (IDA) analysis, demonstrating that IDA was an efficient approach for structural elucidation in metabonomic studies. Citric acid and a glucuronide-containing metabolite were observed as potential biomarkers in rat urine. A significant increase in plasma creatinine concentration was also observed in the AA-dosed rats, which indicated that AA induced an adverse effect on the renal clearance function.

Metabolic activation of carcinogenic aristolochic acid, a risk factor for Balkan endemic nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is associated with tumor development in patients suffering from Chinese herbs nephropathy (now termed aristolochic acid nephropathy, AAN) and may also be a cause for the development of a similar type of nephropathy, the Balkan endemic nephropathy (BEN). Major DNA adducts [7-(deoxyadenosin-N6-yl)-aristolactam and 7-(deoxyguanosin-N2-yl)aristolactam] formed from AA after reductive metabolic activation were found in renal tissues of patients with both diseases. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. This paper reviews major hepatic and renal enzymes responsible for AA-DNA adduct formation in humans. Phase I biotransformation enzymes play a crucial role in the metabolic activation of AA to species forming DNA adducts, while a role of phase II enzymes in this process is questionable. Most of the activation of AA in human hepatic microsomes is mediated by cytochrome P450 (CYP) 1A2 and, to a lower extent, by CYP1A1; NADPH:CYP reductase plays a minor role. In human renal microsomes NADPH:CYP reductase is more effective in AA activation. Prostaglandin H synthase (cyclooxygenase, COX) is another enzyme activating AA in human renal microsomes. Among the cytosolic reductases, NAD(P)H:quinone oxidoreductase (NQO1) is the most efficient in the activation of AA in human liver and kidney. Studies with purified enzymes confirmed the importance of CYPs, NADPH:CYP reductase, COX and NQO1 in the AA activation. The orientation of AA in the active sites of human CYP1A1, -1A2 and NQO1 was predicted from molecular modeling and explains the strong reductive potential of these enzymes for AA detected experimentally. We hypothesized that inter-individual variations in expressions and activities of enzymes activating AA may be one of the causes responsible for the different susceptibilities to this carcinogen reflected in the development of AA-induced nephropathies and associated urothelial cancer.

A sensitivity enhanced high-performance liquid chromatography fluorescence method for the detection of nephrotoxic and carcinogenic aristolochic acid in herbal medicines

A new, sensitive and selective HPLC method with fluorescence detector (HPLC–FLD) for the determination of nephrotoxic and carcinogenic aristolochic acid (AA) in herbal medicines by using pre-column derivatization with zinc powder in acetic acid is presented. Variables governing the derivatization reaction, such as the amount of zinc powder and acetic acid, as well as the derivatization time were studied and optimized. An extended linear dynamic range over three orders of magnitude was observed for AA-I and AA-II ( R 2 > 0.9998). Method accuracy at low, medium and high spiked AA levels determined by the percentage mean deviation was below 4.4% and 7.2% for AA-I and AA-II, respectively. The detection limits of 0.39 ng/mL (AA-I) and 0.52 ng/mL (AA-II) were 2 orders of magnitude lower than those obtained from HPLC-MS or CE-ECD analyses, 3–4 orders of magnitude lower than those from HPLC-UV or CE-UV methods. The developed method has been applied for the determination of AA in herbal medicines. Among the tested samples, Guanmutong had the highest AA concentration (2607.0 μg/g AA-I, 711.2 μg/g AA-II). Comparison studies between HPLC–FLD and HPLC–MS/MS demonstrated that the two methods gave similar quantitative results for the selected herb samples.

Investigation of the Metabolism and Reductive Activation of Carcinogenic Aristolochic Acids in Rats

The metabolic activation of aristolochic acids (AAs) that have been demonstrated to be mutagenic and carcinogenic was investigated. In vitro metabolism study indicated that AAs were metabolized to N-hydroxyaristolactam, which could be either reduced to aristolactams or rearranged to 7-hydroxyaristolactams via the Bamberger rearrangement. In vivo metabolism study is important because the intermediates (aristolactam-nitriumion) of the nitroreduction process are thought to be responsible for the carcinogenicity of AAs. Liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (MS/MS) were applied to the analyses of a series of positional isomers of hydroxyaristolactams in rat urine samples after the in vivo study of AAs. Three hydroxylated metabolites of aristolactam II and two hydroxylated metabolites of aristolactam I were identified. The structures of the positional isomers were elucidated from the interpretation of MS/MS spectra and theoretical calculations. In addition, several new metabolites were detected in the rat urine by high-resolution mass spectrometry and MS/MS, including those from the decarboxylation of AAs and the conjugations of acetylation, glucuronidation, and sulfation of aristolochic acid Ia.

Analysis of nephroloxic and carcinogenic aristolochic acids in Aristolochia plants by capillary electrophoresis with electrochemical detection at a carbon fiber microdisk electrode

Aristolochic acids (AAs) are the main bioactive ingredients in the most of Aristolochia plants, which are used to make dietary supplements, slimming pills and Traditional Chinese Medicines (TCMs). Excessive ingestion of AAs can lead to serious nephropathy. Therefore, quantitative analysis and quality control for the plants containing AAs is of great importance. In this paper, capillary electrophoresis (CE) with electrochemical detection (ED) at a 33 μm carbon fiber microdisk electrode (CFE) has been applied to detect AA-I and AA-II in Aristolochia plants. Under the optimum conditions: detection potential at 1.20 V, 2.0 × 10 −2 mol L −1 phosphate buffer solution (PBS) (pH 10.0), injection time 25 s at a height of 17 cm and separation voltage at 12.5 kV, the AA-I and AA-II were baseline separated within 5 min. Low detection limits for AA-I and AA-II were 4.0 × 10 −8 mol L −1 and 1.0 × 10 −7 mol L −1, respectively. Wide linear ranges were from 4.0 × 10 −8 mol L −1 to 1.9 × 10 −5 mol L −1 and 1.0 × 10 −7 mol L −1 to 5.0 × 10 −5 mol L −1 for AA-I and AA-II, respectively. The proposed method has been successfully applied to analyze AAs contents in plant extracts. The results indicated that the contents of AAs in each part of Aristolochia debilis Sieb. Et Zucc. plant were different. Meanwhile, the CE–ED method was utilized for fingerprint analysis of medicine herbs. Six herbs ( Radix aristolochiae, Fructus aristolochiae, Herba aristolochiae, Caulis aristolochiae manshuriensis, Caulis clematidis armandii, Caulis akebiae) were well distinguished by comparing their electropherograms obtained by CE–ED method.

Detection of aristolochic acid I, tetrandrine and fangchinoline in medicinal plants by high performance liquid chromatography and liquid chromatography/mass spectrometry

Problems with identification and labeling of medicinal plants, as well as substitution/adulteration of non-toxic plants by toxic ones have previously led to cancer, renal failure and even deaths. The non-toxic Stephania tetrandra (Fangji) has been known to be substituted by Aristolochia fangchi (Guang fangji), which contains the nephrotoxic and carcinogenic aristolochic acid (AA). In this study, 10 samples of “Fangji” were bought from local medicinal shops. HPLC-DAD chromatographic fingerprints of each methanol extract were compared with those of A. fangchi and S. tetrandra, using aristolochic acid I (AAI), tetrandrine and fangchinoline as marker compounds. Nine of the samples were found to be similar to A. fangchi. The presence of AAI in the nine samples was confirmed using LC-MS/MS. Neither tetrandrine nor fangchinoline were detected in these samples. The methods developed in this study allow the simultaneous detection of AAI, fangchinoline and tetrandrine. The results suggest possible substitution of S. tetrandra by A. fangchi at wholesale or retail level. This study highlights the importance of greater control of medicinal plants with toxic components as these may still be readily accessible to the public.